Background: In vitro maturation of human primary myoblasts using 2D culture remains a challenging process and leads to immature fibers with poor internal organization and function. This would however represent a valuable system to study muscle physiology or pathophysiology from patient myoblasts, at a single cell level. Methods: Human primary myoblasts were cultured on 800 nm width striated surface between two layers of Matrigel, and in a media supplemented with an inhibitor of TGFreceptor. Gene expression, immunofluorescence and Ca2+ measurements upon electrical stimulations were performed at various time points during maturation to assess the organization and function of the myotubes. Results: We show that after 10 days in culture, myotubes display numerous functional acetylcholine receptor clusters and express the adult isoforms of myosin heavy chain and dihydropyridine receptor. In addition, the myotubes are internally well organized with striations of -actinin and STIM1, and occasionally ryanodine receptor 1. We also demonstrate that the myotubes present robust Ca2+ responses to repetitive electrical stimulations. Conclusion: The present method describes a fast and efficient system to obtain well matured and functional myotubes in 2D culture allowing thorough analysis of single cell Ca2+ signals.